Current standard-of-care antiviral regimens rely on direct-acting antivirals (DAAs) possessing inherent liabilities. DAAs are generally active against only one virus or a closely related, family of viruses. Because a viral protein is targeted, the virus can readily develop resistance mutations. To address the shortcomings of DAAs, FORGE Life Science is developing host-targeted antivirals (HTAs). HTAs have the potential to block the growth of multiple different viruses. Since a host-cell protein is targeted, viruses are much less likely to evolve drug resistance. Specifically, FORGE is developing small molecule drugs that target human sirtuin proteins. Sirtuins are a family of seven protein-deacylases that modulate many cellular processes critical for virus replication. This proposal seeks to develop a sirtuin-modulating drug that is simultaneously effective against multiple different opportunistic viruses causing life-threatening disease in immunosuppressed transplant patients. Initially, the program is focused on human cytomegalovirus (HCMV). In Phase 1, a chemical series of uncompetitive sirtuin 2 (SIRT2) inhibitors was developed that block the production of HCMV progeny in cultured human cells more potently than standard-of-care, ganciclovir. Strikingly, these SIRT2 inhibitors not only affect HCMV, but they also inhibited the growth of influenza A and B, hepatitis B and C viruses, and the polyomaviruses, BKV and JCV. The compound series demonstrates structure activity relationship to antiviral potency, excellent oral bioavailability, and good tolerability in mice. In vivo validation of anti-HCMV activity was achieved in immunosuppressed mice carrying human lung-tissue implants. This application proposes to move the program forward to SBIR Phase II to optimize a development candidate for progression into preclinical development. Three aims will be pursued. (1) A mouse model supporting development of recently approved DAA letermovir will be adapted for use with SIRT2 inhibitors. This model allows for HCMV infection of human fibroblasts seeded to a Gelfoam implant placed into immunodeficient SCID mice. (2) A medicinal chemistry campaign will refine the current lead SIRT2 inhibitor to improve anti-HCMV activity in the Gelfoam/SCID mouse with a target to achieve a therapeutic index equivalent or better than oral-dosing valganciclovir and letermovir. The selected development candidate will additionally satisfy in vitro ADME and pharmaceutical criteria, including minimizing drug-drug interactions, for administration to transplant patients. (3) Compounds demonstrating sufficient efficacy in the Gelfoam/SCID mouse model will be validated with respect to providing a high barrier to acquired viral drug-resistance and to synergize with existing direct-acting antivirals. The pan-viral profile will be expanded to multiple opportunistic viruses. This project has the potential to produce a paradigm shift, introducing broad-spectrum antivirals that solve the problem of viral resistance.